Date Published: September 12, 2012
Publisher: Public Library of Science
Author(s): Carola Maffioli, Denis Grandgirard, Stephen L. Leib, Olivier Engler, Pedro Lagerblad Oliveira. http://doi.org/10.1371/journal.pone.0044703
Tick-borne encephalitis virus is the causative agent of tick-borne encephalitis, a potentially fatal neurological infection. Tick-borne encephalitis virus belongs to the family of flaviviruses and is transmitted by infected ticks. Despite the availability of vaccines, approximately 2000–3000 cases of tick-borne encephalitis occur annually in Europe for which no curative therapy is available. The antiviral effects of RNA mediated interference by small interfering RNA (siRNA) was evaluated in cell culture and organotypic hippocampal cultures. Langat virus, a flavivirus highly related to Tick-borne encephalitis virus exhibits low pathogenicity for humans but retains neurovirulence for rodents. Langat virus was used for the establishment of an in vitro model of tick-borne encephalitis. We analyzed the efficacy of 19 siRNA sequences targeting different regions of the Langat genome to inhibit virus replication in the two in vitro systems. The most efficient suppression of virus replication was achieved by siRNA sequences targeting structural genes and the 3′ untranslated region. When siRNA was administered to HeLa cells before the infection with Langat virus, a 96.5% reduction of viral RNA and more than 98% reduction of infectious virus particles was observed on day 6 post infection, while treatment after infection decreased the viral replication by more than 98%. In organotypic hippocampal cultures the replication of Langat virus was reduced by 99.7% by siRNA sequence D3. Organotypic hippocampal cultures represent a suitable in vitro model to investigate neuronal infection mechanisms and treatment strategies in a preserved three-dimensional tissue architecture. Our results demonstrate that siRNA is an efficient approach to limit Langat virus replication in vitro.
Tick-borne encephalitis virus complex represents a group of closely related viruses endemic in Europe and Asia causing serious neuroinfections and hemorrhagic fevers . Tick-borne encephalitis virus (TBEV) belong to the Flaiviridae family and can be divided in three main subtypes, the European, the Siberian and Far Eastern subtypes . TBEV are transmitted to humans by the bite of infected ticks and, in rare cases, through the consumption of infected unpasteurized milk , . Following an incubation period of 3–8 days after a tick bite, the virus replicates locally in epidermal dendritic cells and spreads via lymph vessels to the blood stream where a short but significant viremia occurs and extraneural tissues are infected. It is during this viremic phase that the virus crosses the blood-brain barrier and invades the central nervous system (CNS) by a still unknown mechanism. Once the virus has reached the CNS, active infection causes inflammation, lysis of cells and cellular dysfunction , . Clinical manifestations of TBEV infections typically follow a biphasic course. The first viremic phase is characterized by flu-like symptoms such as fever, headache and muscle pain. After 3–7 days of an asymptomatic phase, 20–30% of the patients develop the second meningoencephalitic phase with neurological disorders of varying severity , , , . The most common clinical feature of TBE patients is ataxia followed by paresis or paralysis of one or more extremities .
Therapeutic options for the treatment of TBE are lacking and specific therapies are urgently needed since the incidence of TBE is rising. Several studies have shown that siRNA molecules have the potential to be used as a specific therapeutic strategy against viral infections. Due to difficulties in delivery, toxicity and the stimulation of unspecific immune response few approaches were continued in vivo. For TBE virus infections detailed investigations determining optimal target sequence and inhibitory capacity for synthetic siRNA are lacking. The majority (11/19) of the 19 siRNA sequences screened in this study were capable of reducing Langat virus titer by more than 80% in comparison to nonsense siRNA when transfected into the cells before the infection (Figure 1 B). The most effective reduction in viral replication was achieved with siRNA sequences targeting the 3′ UTR and the structural genes. SiRNA sequence D3 (5′UTR) and Q6 (Envelope) showed the highest capacity to inhibit viral replication, reducing the number of viral RNA genome copies from 1×10E6/µl (nonsense siRNA) to 5.5×10E4/µl (siRNA D3) and 6.6×10E4/µl (siRNA Q6) respectively, corresponding to a reduction of more than 93% (Figure 1 B). In addition to its strong antiviral activity sequence D3 is highly conserved between different members of the tick-borne encephalitis virus complex and was therefore selected for further experiments on HeLa cells and organotypic brain cultures. Using the siRNA sequence D3 we could demonstrate that the application of siRNA after the infection was still effective. In fact the inhibitory effect was comparable to the results obtained when siRNA was applied before infection. These results indicate that it is feasible, at least in the paradigm tested herein, to inhibit replication of TBEV in vitro once the infection has been initiated.